The overall objectives of this project are to clarify the molecular mechanisms by which inducible nitric oxide synthase (iNOS) mediates insulin resistance. The previous studies revealed that: (1) iNOS inhibition significantly ameliorates whole-body insulin resistance, and improves depressed expression of insulin receptor substrate (IRS)-1 and IRS-2, and IRSs-mediated insulin signaling in obese, diabetic (ob/ob) mice;(2) S-nitrosylation reversibly inactivates Akt/PKB;and that (3) S-nitrosylated Akt/PKB was increased in diabetic mice. Chronic inflammation and cellular stress signaling have been implicated in obesity-induced insulin resistance. However, its origin and potential cross-talks between the stress signaling pathways remain elusive. The preliminary experiments showed that: (1) gene disruption of S-nitrosoglutathione reductase (GSNOR), a negative regulator of protein S-nitrosylation, caused insulin resistance and aggravated high-fat diet-induced diabetes in mice;(2) activation of endoplasmic reticulum (ER) stress, NF- kappaB and c-Jun N-terminal kinase (JNK/SAPK) was attenuated by iNOS inhibitor in the liver of ob/ob mice;and (3) alleviation of ER stress by overexpression of an ER chaperone blocked iNOS-induced decrease in IRS-2 expression in cultured hepatocytes. Based on the previous studies and convincing preliminary data, the following hypotheses will be tested: that protein S-nitrosylation plays an important role in insulin resistance;and that iNOS contributes to the development of insulin resistance not only as a downstream effector of inflammation, but also as an enhancer of stress signaling pathways. Aim 1 will determine a role of GSNOR in insulin resistance in GSNOR knockout mice and liver-specific GSNOR transgenic mice. Aim 2 will determine whether increased iNOS expression in liver is sufficient to induce insulin resistance in mice. Aim 3 will clarify a role of iNOS in activation of ER stress, IKK-NF-kappaB and JNK/SAPK pathways in ob/ob mice. Aim 4 will clarify a role of ER stress in iNOS-mediated insulin resistance. The proposed studies will provide novel, significant insights to understand the pathogenesis of insulin resistance in an integrated manner. Insulin resistance, attenuated responsiveness to insulin, is a major causative factor for diabetes, and a risk factor for ischemic heart disease. This project will provide new information to establish novel strategies to prevent and/or treat insulin resistance and obesity-related diabetes.